Staphylokinase, an extracellular protein of several strains of Staphylococcus aureus, is a promising blood-clot dissolving agent that carries many desirable thrombolytic properties and is useful for the treatment of patients suffering from thromboembolic disorders and myocardial infarcation [Collen et. al., Fibrinolysis, vol. 6; 232-242, 1992; Collen et. al., vol. 87, 996-1006, 1993]. It forms a bimolecular complex with the blood proteins, such as plasminogen (PG) and plasmin (Pm) and exerts its fibrinolytic effects through conversion of an active non specific serine protease, plasmin (Pm) to a highly specific proteolytic enzyme that can recognize blood zymogen, PG, as a substrate and convert it into plasmin that is capable of degrading blood clots. In a plasma milieu, SAK, is able to dissolve fibrin clots without any associated fibrinogen degradation [Collen et. al., J. Biol. Chem. 268, 8284-8289, 1993; Lijnen et. al., J. Biol. Chem. 266, 11826-11823]. This fibrin-specificity of SAK is the result of inhibition of staphylokinase-plasmin complex formation by alpha-2-antiplasmin during circulation in blood but highly diminished interaction with staphylokinase-plasmin complex at the fibrin surface, resulting in very localized plasminogen activation at the fibrin surface. In addition, staphylokinase has weak affinity for circulating plasminogen but a high affinity for fibrin-bound plasminogen. Recent clinical trials have shown that staphylokinase is as effective as t-PA at achieving early perfusion in myocardial infarction patients. Thus, its utility in thrombolytic treatment has now been established by several limited clinical trials [Collen et. al., Circulation, 87, 996-1006, 1993; Lijnen and Collen, Fibrinolysis, 10, 119-126, 1996].
Staphylokinase is a single chain 16 kD protein, consisting of 136 amino-acid residues It is produced in very low amounts by its natural host, Staphylococcus aureus [Lack, Nature 161, 558-560, 1948; Robbinson et. al., J. Biol. Chem. 242, 2333-2342, 1967]. Production of intact, biologically active SAK from bacterial expression systems has been a challenge because of N-terminal micro-heterogeneity, plasmid instability, or low-production yield. Considering its therapeutic applicability and clinical implications in thrombolytic therapy, attempts have been made in the past (Gerlach et. al., Zbl. Bakt. Hyg. A269, 1988; Collen et. al., Fibrinolysis 6, 203-213, 1992) to search for an alternative source of SAK production through recombinant routes. The gene encoding for SAK has been isolated from its natural host, Staphylococcus aureus, and cloned into various heterologous hosts, e.g., E. coli, Bacillus and Yeast [Sako et. al., Mol. Gen. Genet., 190, 271-277, 1983]. In the case of Bacillus SAK appeared proteolytically degraded [Ruiqiong et. al., Biotechnol. Bioeng., 62, 87-96, 1998; Miele, et. al., J. Biol. Chem. 274, 7769-7776, 1999] and in Yeast it was found glycosylated that reduced the plasminogen-activation function of this protein. Therefore, these heterologous systems were not very suitable for the large scale production of this protein. In E. coli, extracellular production of SAK resulted in low level of SAK production carrying N-terminal degradation resulting in two forms of SAK. To overcome these problems, SAK encoding gene has been expressed using strong expression signals which required addition of exogenous inducers, such as IPTG, tryptophane, Indol-acetic acid etc. that makes these systems highly expensive when utilized for the large scale production of this protein. In this system, production of full length SAK was associated with a truncated form of SAK that lacked 10-N terminal amino acid residues of native SAK.
The present invention, therefore, is concerned with a new method for the high yield production of staphylokinase and its analogs using a new protein expression signal that does not require use of any expensive exogenous chemicals to induce the protein production and therefore provides an economic advantage over the currently known procedure for the production of staphylokinase. Moreover, the staphylokinase and its derivatives produced via this process display specific clot lysis activity and human plasminogen activation capability that is comparable to its native counterpart. Thus, in principle, the present process constitutes a new and more economical means for the production of staphylokinase and its derivatives that may be useful in thrombolytic therapy.